BS/2009/062

Experiment No. 03

CHEM 43272

Date Experiment No. Title

: 23.09.2013 : 03 : Enzyme kinetics

Objectives

: To emphasize the principles of enzyme kinetics and how to conduct enzyme assays. To determine the Km and Vmax for tyrosinase isolated from mushrooms

Abstract :

Tyrosinase is a copper-containing oxidoreductase enzyme that can be found in plant and animal tissues that catalyzes the oxidation of tyrosine into melanin and other pigments. Tyrosinase was extracted from edible mushroom (Agaricus sp.) and the kinetic parameters of the extracted enzyme was determined by the catalytic oxidation of catechol using a spectrophotometric method. The molar extinction coefficient for the oxidized product was determined to be 19,400 mol-1 dm-3 cm-1. The Observed Km & Vmax values using the Michelis-Menton plot are 0.4 mM and 0.030 mol min-1 & the Observed Km & Vmax values using the Lineweaver-Burk plot were 2.00 mM & 0.10 mol min-1.

Introduction : Enzymes are biological catalysts. Without their presence in a cell, most bio chemical reactions would not proceed at the required rate. The physicochemical and biological properties of enzymes have been investigated since the early 1800 s. The unrelenting interest in enzymes is due to several factors, their dynamic and essential role in the cell, their extraordinary catalytic power, and their selectivity. Two of these dynamic characteristics will be evaluated in this experiment, namely a kinetic description of enzyme activity and molecular selectivity. Enzyme catalyzed reactions proceed through an ES complex as shown in Equation below. The individual rate constants, kn, are placed with each arrow.

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Experiment No. 03

CHEM 43272

E represents the enzyme, S the substrate or reactant, and P the product. For a specic enzyme, only one or a few different substrate molecules can bind in the proper manner and produce a functional ES complex. The substrate must have a size, shape, and polarity compatible with the active site of the enzyme. Some enzymes catalyze the transformation of many different molecules as long as there is a common type of chemical linkage in the substrate. Others have absolute specificity and can form reactive ES complexes with only one molecular structure. In fact, some enzymes are able to differentiate between D and L isomers of substrates.

The initial reaction velocity, Vo, of an enzyme-catalyzed reaction varies with the substrate concentration, [S], as shown in above. The Michaelis-Menten equation has been derived to account for the kinetic properties of enzymes. (Consult a biochemistry textbook for a derivation of this equation and for a discussion of the conditions under which the equation is valid.)

The common form of the equation is:

where Vo Vmax = initial reaction velocity = maximal reaction velocity; attained when all enzyme active sites are lled with substrate molecules = substrate concentration = Michaelis constant

[S] KM

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The important kinetic constants, Vmax and KM can be graphically determined as shown in above graph. Equation stated above and above graph have all of the disadvantages of nonlinear kinetic analysis. Vmax can be estimated only because of the asymptotic nature of the line. The value of KM, the substrate concentration that results in a reaction velocity of Vmax / 2, depends on Vmax, so both are in error. By taking the reciprocal of both sides of the Michaelis-Menten equation, however, it is converted into the LineweaverBurk relationship.

This equation, which is in the form y = mx + c , gives a straight line when 1/Vo is plotted against 1/[S] The intercept on the 1 / Vo axis is 1/Vmax and the intercept on the 1/[S] axis is 1/KM. A disadvantage of the Lineweaver-Burk plot is that the data points are compressed in the high substrate concentration region.

BS/2009/062

Experiment No. 03

CHEM 43272

BS/2009/062

Experiment No. 03

CHEM 43272

Materials : UV-VIS Spectrophotometer Quartz cuvettes Glass pipettes Pipette filer Micro pipette Test tubes Measuring cylinder Volumetric flasks Electrical balance Analytical balance pH meter Spatula Watch glass Beakers Motor and Pestle Mushrooms Muslin cloth Centrifuger Centrifuge tubes Tissues Na2HPO4.2H2O(s) Conc. H3PO4 Catechol(s) Tyrosinase enzyme solution (1 mg/mL) Distilled water

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Procedure : Phosphate buffer (pH 6.5, 0.05 M, 250.00 mL) was prepared using Na2HPO4.2H2O(s) (2.27 g) and addition of sufficient amount of the Conc. H3PO4. Mushroom (50..00 g) was accurately weighed and the tyrosinase enzyme was extracted using the pH 6.5 buffer solution using the motor and pestle. Extracted solution was separated from the cell debrides by filtering using an muslin cloth. Filtered solution was centrifuged at 2500 rpm to precipitate any solid material. Catechol solution (20 mM, 100.00 mL) was prepared using catechol(s) (0.2203 g). Then a series of catechol solutions were prepared according to the following table.

Tube #

20 mM Catechol volume/ mL 0.10 0.50 1.00 2.00 2.50 3.00 4.00 5.00

Buffer volume / mL

Catechol concentration / mM 0.20 1.00 2.00 4.00 5.00 6.00 8.00 10.00

1 2 3 4 5 6 7 8

8.90 8.50 8.00 7.00 6.50 6.00 5.00 4.00

Solutions were taken (one at a time) and the extracted enzyme solution (1.00 mL) was added and the reaction was monitored by taking the absorbance values in every 30 second time interval up to 3 minutes from the starting time at 490 nm using the buffer solution as the blank. A solution of catechol (0.2 mM, 100.00 mL) was prepared using catechol(s)(0.0022 g) dissolving in distilled water and a series of dilutions were prepared according to following table. Tube # 0.02 mM Catechol volume/ mL 0.25 1.00 2.50 4.00 5.00 Buffer volume / mL Dilution 8.75 8.00 6.50 5.00 4.00 1:4 1:4 1:4 1:4 1:4 Catechol concentration / mM 0.001 0.004 0.010 0.016 0.020

1 2 3 4 5

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Extracted enzyme solution (1.00 mL) was added to each solution and allowed to complete the reaction for an hour and the absorbances of the final solutions were measured at 490 nm. A graph was plotted , absorbance at 490 nm vs. the catechol concentration and the molar absorption coefficient was calculated according to the graph. The rate of the reaction was determined (mol/min) using the observed value for and using the absorbance values obtained from the initial table. A graph was plotted , the rate of the reaction vs. substrate concentration and the Vmax and KM values were calculated according to that graph. Obtained values were compared with the reported values. Another linear graph was plotted according to the Lineweaver and Burk equation and the Vmax and KM values were calculated according to that graph. The values obtained for both methods were compared.

Results : Determining the for the product.

Tube #

Absorbance at 490 nm

0.219

2 3

0.226 0.256

0.273

0.349

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Rate determination

Solution umber 1

Time / min 0.0 1.5 2.0 3.0 4.5 5.0 0.0 0.5 1.0 2.0 3.0 0.0 0.5 1.0 2.0 2.5 3.0 0.0 1.0 2.0 3.0 0.5 1.0 2.0 2.5 3.5 0.5 1.5 2.0 3.0

Absorbance at 490 nm 0.247 0.333 0.508 0.540 0.582 0.633 0.351 0.612 0.656 0.664 0.672 0.420 0.460 0.477 0.512 0.552 0.700 0.224 0.254 0.278 0.329 0.287 0.293 0.311 0.347 0.349 0.173 0.250 0.269 0.281

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Experiment No. 03

CHEM 43272

Solution umber 7

Time / min 1.0 2.0 3.0 0.5 1.5 2.5 3.0

Absorbance at 490 nm 0.415 0.644 0.724 0.179 0.189 0.204 0.240

Analysis and calculations :

Catechol concentration (Product concentration) / M 1.0

Absorbance at 490 nm

0.219

4.0

0.226

10.0

0.256

16.0 20.0

0.273 0.349

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The graph of absorbance at 490 nm Vs. the concentration of the product / M

Absorbance at 490 nm 0.45

0.40

0.35

y = 0.0194x

0.30

0.25

0.20

0.15

0.10

0.05

0.00 0.0 5.0 10.0 15.0 20.0 25.0 Concentration of the product / M

The gradient of the graph = 0.0194 x l = 0.0194 ( in M units) But l = 1 cm = 19400 mol- dm3 cm-1 (1.94 x 104 mol- dm3 cm-1)
10

at 490 nm = 0.0194 x 106 mol- dm3 cm-1

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Experiment No. 03

CHEM 43272

Rate determination i.e. solution number 1

2cathecol (1,2-dihydroxybenzene) + O 2 ortho-quinone + 2 H O
2 2

According to the equation the rate of substrate reacting is equal to the rate formation of product.

Absorbance at t ,0.00 min concentration at 0.00 min Amount of product in 10.00 mL

= 0.247 = 0.0247 1.94 x 104 mol- dm3 cm-1 = 0.247 x 4 1.94 x 10 mol dm-3

10.00 mL x 106 mol 1000.00 mL

= 0.127 mol At time, 5.00 min amount of product in 10.00 mL = 0.633 1.94 x 104 = 0.326 mol Rate of the reaction = (0.326 - 0.127) / 5.0 min = 0.040 mol min-1 x 10.00 mL x 106 mol 1000.00 mL

Solution umber 1

Time / min 0.0 1.5 2.0 3.0 4.5 5.0 0.0 0.5 1.0 2.0 3.0

Product amount in 10.00 mL / mol 0.127 0.172 0.262 0.278 0.300 0.326 0.181 0.315 0.338 0.342 0.346
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Rate of the reaction 0.040

0.055

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CHEM 43272

Solution umber 3

Time / min 0.0 0.5 1.0 2.0 2.5 3.0

Product amount in 10.00 mL / mol 0.216 0.237 0.246 0.264 0.285 0.361 0.115 0.131 0.143 0.170 0.148 0.151 0.160 0.179 0.180 0.089 0.129 0.139 0.145

Rate of the reaction 0.048

0.0 1.0 2.0 3.0

0.018

0.5 1.0 2.0 2.5 3.5

0.0107

0.5 1.5 2.0 3.0

0.022

1.0 2.0 3.0

0.214 0.332 0.373 0.092 0.097 0.105 0.124

0.080

0.5 1.5 2.5 3.0

0.013

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CHEM 43272

Substrate concentration, [S] / mM 0.20 1.00 2.00 4.00 5.00 6.00 8.00 10.00

Rate of the reaction / mol min-1 0.040 0.055 0.048 0.018 0.011 0.022 0.080 0.013

The graph of rate / mol min-1 Vs. substrate concentration, [S] / mM

rate / mol min-1 0.090

0.080

0.070

Vmax
0.060

0.050

0.040

Vmax / 2
0.030

0.020

0.010

KM

0.000 0.00 2.00 4.00 6.00 8.00 10.00 12.00 [S] / mM

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Experiment No. 03

CHEM 43272

According to the observed graph Vmax for the tyrosinase KM for the tyrosinase = 0.030 mol min-1 = 0.4 mM

Formation of the Lineweaver-Burk plot

1 / [S] (mM-1) 5.00 1.00 0.50 0.25 0.20 0.17 0.13 0.10

1 / V (mol-1 min) 25.00 18.18 20.83 55.56 90.91 45.45 12.50 76.92

According to the observed graph, 1/ Vmax Vmax = 10.00 mol-1 min = 0.10 mol min-1

-1 / KM KM

= - 0.50 = 2.00 mM

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The graph of 1 / [S] Vs. 1 / V

1/V 100.00

90.00

80.00

70.00

60.00

50.00

40.00

30.00

20.00

10.00

0.00 -3.00 -2.00 -1.00 0.00 1.00 2.00 3.00 4.00 5.00 6.00

1 / [S]

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Experiment No. 03

CHEM 43272

Discussion : The KM and the Vmax values for an particular enzyme changes with lot of parameters. pH of the medium, temperature highly affect to the activity of the enzyme as well as for the kinetic assay. The KM and the Vmax for a particular enzyme also changes with the substrate and the source of the enzyme. Literature says that the optimum pH for the mushroom tyrosinase(from Agaricus species) is 6 and also the optimum temperature is 27 oC.

Observed values for the absorbance of the product showed some fluctuations. Therefore the concentrations and also the rate values also showed confusing fluctuations when plotted to the graphs. Both Michelis-Menton and Lineweaver-Burk plots showed lot of confusing data distributions that not to give correct graph form for the theoretical equations. All these observations should be due to some error of the experimental design or due to the instrumental error that was used. When substrate concentration is increased, the product concentration also should be increased thus giving an increase of the absorbances. But the observation was for lot of concentrations was decrease of the absorbances. So there should be an instrumental error for these observations.

The calculated KM and the Vmax values from the Michelis-Menton curve were 0.4 mM and 0.030 mol min-1. Literature says these values for the tyrosinase when using catechol as the substrate at 27 o C and 0.1 M phosphate buffer as 0.60 mM and 0.38 mol min-1. So the observed values are much more deviated from those values. Calculated KM and the Vmax values from the Lineweaver-Burk plot were 2.00 mM & 0.10 mol min-1. Even these values were not close for the literature values. He values obtained from two methods also showed much more deviation from each other. When looking at the two graphs they clearly show the deviations from the theoretical equations. So this method should be improved to gain correct results using a reliable range of concentrations of the substrate. A reliable spectrophotometer should be used to measure the absorbances. A purification step for the extracted enzyme is necessary to concentrate the enzyme. Testing the activity of the enzyme before proceed the kinetic assay is also good practice. These improvements will lead to obtain reliable results for the kinetic assay of this enzyme.

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Experiment No. 03

CHEM 43272

Tyrosinase catalyzes the oxidation of catechol to produce ortho-quinone. Quinone is any member of a class of diketones derivable from aromatic compounds by conversion of two CH groups into CO groups. Quinones form a large, varied and widespread group of compounds; they include pigments, electron carriers and vitamins K. The molar ratio of the substrate and the product is 1 : 1. Therefore the reaction rate of the substrate is equal to reaction rate of the product. The product ortho-quinone gives absorption maxima at 490 nm that can be determined colorimetrically. (using an spectrophotometer.). Instead of finding the rate of the reaction for mol min-1 units, the absorption variation rate also can be measures as the absorbance directly proportional to the concentration. As we are dealing with the mol and mmol concentrations the colored compound should contain larger molar extinction coefficient to give a absorbance reading even at low concentrations. The determined for the product was 19400 mol- dm3 cm-1 that gives spectrometrically measurable absorbance value in low concentrations.

Conclusions : Observed Km & Vmax values using the Michelis-Menton plot are 0.4 mM and 0.030 mol min-1 Observed Km & Vmax values using the Lineweaver-Burk plot were 2.00 mM & 0.10 mol min-1

References : Rodney Boyer, 2000, Modern experimental Biochemistry, 3 rd edition, Benjamin Cummings, San Francisco, CA, pp 279 - 300. http://198.170.104.138/biotech/2006/344-348.pdf [28.09.2013]

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